本研究利用海藻酸鈉與明膠於交聯前易於塑型之特性，研究兩者作為骨缺損患部原位注射型骨填補材之可行性。利用明膠具有溫度敏感之特性，將鈣離子摻雜其中，作為鈣離子釋放載體，以延長海藻酸鈉與鈣離子完全交聯之時間，並研究細胞封裝，以避免交聯反應發生時對細胞造成之損害，及一同以注射方式植入患部之可行性。以流變儀 (rheometer)與示差掃描量熱儀 (differential scanning calorimeter, DSC)以2 ℃/min的速率自4 ℃開始升溫，量測鈣離子摻入明膠載體後，對熔融溫度 (melting temperature, Tm)之影響。以穿透式電子顯微鏡 (transmission electron microscope, TEM)觀測添加檸檬酸對於實驗鈣離子來源之一的氫氧基磷灰石 (hydroxyapatite, HAp)助分散之效果。以流變儀 (rheometer)測定海藻酸鈉支架交聯過程及支架受溫度影響機械性質之變化。生物相容性部分，以骨母細胞hFOB 1.19細胞株作為患部細胞之代表，纖維母細胞L-929細胞株代表周圍組織細胞，依ISO 10993-5之規範，以待測樣品依培養液萃取物進行體外細胞毒性測試，並以MTT assay的方式將結果量化；將細胞封裝前後之兩細胞株分別植入 (seeding)支架，觀察並比較細胞增生之情形。觀察支架對骨母細胞hFOB 1.19細胞株生物活性包括鹼性磷酸酶活性 (alkaline phosphatase activity, ALPase activity)、蛋白質生成之影響。研究發現，氯化鈣摻入明膠之中，會使明膠熔融溫度下降，且加入之氯化鈣越多，熔融溫度下降越明顯；HAp雖不影響明膠之熔融溫度，但用來做為助溶劑之檸檬酸易會造成明膠熔融溫度下降，檸檬酸濃度越高，明膠熔融溫度下降越明顯。透過TEM觀測，2.5 %檸檬酸之添加，有效將原先直徑約2μm之HAp分散為直徑100 nm之大小。由流變儀測試結果可見，含鈣明膠載體與海藻酸鈉接觸後，因海藻酸鈉與鈣離子發生交聯反應使材料彈性增加，最終材料穩定彈性會隨含鈣明膠載體添加量而增加；而以明膠作為鈣離子釋放載體，也有效的使海藻酸鈉交聯完全的時間延長至15分鐘左右，有利於應用時的加工與塑型。細胞毒性測試發現，未與海藻酸鈉發生交聯之含HAp明膠萃取物似乎不利於L-929之正常生長，而hFOB 1.19細胞株似乎對環境適應性較差，所有樣品萃取物造成了部分細胞死亡，但存活者隨時間增加，仍有增長趨勢。明膠細胞封裝載體有利於細胞於不固定介面之附著，但封裝一過程卻對hFOB 1.19細胞株之生存有不利的影響。支架對骨母細胞hFOB 1.19細胞株鹼性磷酸酶活性、蛋白質生成皆有正面的影響，顯示可促進hFOB 1.19進行初步分化，具有發展成為骨組織工程支架的潛力。

An in situ injectable hydrogel made of sodium alginate and gelatin was prepared to study the feasibility for bone tissue engineering. Base on the temperature-sensitivity of gelatin, which was loaded with calcium ions, the time of sodium alginate was extended until reaching body temperature. In addition, cells were encapsulated with gelatin to avoid the damage during gelation and to facilitate the cells by injection into the bone defect. Rheometer and differential scanning calorimeter (DSC) were employed to determine the effect of doping the calcium ion in gelatin on the melting temperature (Tm) of gelatin. Transmission electron microscope (TEM) was used to determine the effect of citric acid as dispersant for hydroxyapatite (HAp). Rheometer was also employed to detect the gelation of alginate scaffold via temperature-scanning. The biocompatibility of scaffold was evaluated with osteoblast (hFOB 1.19) as the representative of the affected area, fibroblasts (L-929) as the representative of the surrounding tissue. According to ISO 10993-5, the in vitro cytotoxicity test was based on culturing cells with the extract of test samples. The effect of encapsulation was investigated by the proliferation of encapsulated cells and free cells on the scaffold. The functionality of osteoblast was observed by the alkaline phosphatase (ALPase) activity and protein production. The results showed that by mixing calcium chloride, Tm of gelatin was decreased with the increase of the content of calcium chloride. The presence of HAp didn’t affect the Tm of gelatin. However, by using citric acid as the dispersant, the Tm of gelatin decreased with the concentration of citric acid. The TEM micrographs showed that the size of HAp was reduced from 2μm to 100 nm by 2.5 % citric acid. The result of rheometer show that when calcium-loading gelatin beads contacted with sodium alginate, the elasticity of the gel increased with the ratio of calcium loading gelatin. Thus using the gelatin as the calcium ions carrier can effectively extend the gelation time of alginate by 15 min, which would be useful for processing and shaping when administration. The in vitro cytotoxicity test showed that the extract of HAp-loading gelatin beads without crosslinking with alginate seemed not suitable for L-929 to grow. Furthermore, hFOB 1.19 seemed sensitive to the environment: although the samples extracts caused death to part of cells, the survivors could still grow with time. The cell encapsulated carrier is good for cell to adhere on the unstable surface, but the process of encapsulation is not good for hFOB 1.19 to survive. The hFOB 1.19 growing on the scaffold can express ALPase activity and produce proteins, suggesting this scaffold can promote the initial differentiation of hFOB 1.19. Thus the scaffolds have the potential to become the bone tissue engineering scaffolds.

1.Swetha, M., et al., Biocomposites containing natural polymers and hydroxyapatite for bone tissue engineering. International Journal of Biological Macromolecules, 2010. 47(1): p. 1-4.
2.Murugan, R. and S. Ramakrishna, Bioresorbable composite bone paste using polysaccharide based nano hydroxyapatite. Biomaterials, 2004. 25(17): p. 3829-3835.
3.Yeatts, A.B. and J.P. Fisher, Bone tissue engineering bioreactors: Dynamic culture and the influence of shear stress. Bone, 2011. 48(2): p. 171-181.
4.Haroun, A.A. and V. Migonney, Synthesis and in vitro evaluation of gelatin/hydroxyapatite graft copolymers to form bionanocomposites. Int J Biol Macromol, 2010. 46(3): p. 310-6.
5.Murugan, R. and S. Ramakrishna, Development of nanocomposites for bone grafting. Composites Science and Technology, 2005. 65(15-16): p. 2385-2406.
6.Matsuno, T., et al., Alveolar bone tissue engineering using composite scaffolds for drug delivery. Japanese Dental Science Review, 2010. 46(2): p. 188-192.
7.Hunt, N.C. and L.M. Grover, Cell encapsulation using biopolymer gels for regenerative medicine. Biotechnol Lett, 2010. 32(6): p. 733-42.
8.Chung, Y.S., et al., Preparation of hydroxyapatite/poly(vinyl alcohol) composite film with uniformly dispersed hydroxyapatite particles using citric acid. Journal of Applied Polymer Science, 2007. 104(5): p. 3240-3244.
9.Lian, J.B., et al., Osteocalcin gene promoter: unlocking the secrets for regulation of osteoblast growth and differentiation. J Cell Biochem Suppl, 1998. 30-31: p. 62-72.
10.李政昕, 脂肪組織中獲取具全能性幹細胞：應用在聚乳酸－聚乙醇酸共聚物三度立體支架之骨組織工程, in 生物科技研究所2002, 國立成功大學: 台南市. p. 120.
11.Curcio, M., et al., Grafted thermo-responsive gelatin microspheres as delivery systems in triggered drug release. European Journal of Pharmaceutics and Biopharmaceutics, 2010. 76(1): p. 48-55.
12.Lau, T.T., C. Wang, and D.-A. Wang, Cell delivery with genipin crosslinked gelatin microspheres in hydrogel/microcarrier composite. Composites Science and Technology, 2010. 70(13): p. 1909-1914.
13.Sakai, S., et al., An injectable, in situ enzymatically gellable, gelatin derivative for drug delivery and tissue engineering. Biomaterials, 2009. 30(20): p. 3371-7.
14.Wang, F., et al., Injectable, rapid gelling and highly flexible hydrogel composites as growth factor and cell carriers. Acta Biomaterialia, 2010. 6(6): p. 1978-1991.
15.Hori, Y., A.M. Winans, and D.J. Irvine, Modular injectable matrices based on alginate solution/microsphere mixtures that gel in situ and co-deliver immunomodulatory factors. Acta Biomaterialia, 2009. 5(4): p. 969-982.
16.Takei, T., et al., In situ gellable sugar beet pectin via enzyme-catalyzed coupling reaction of feruloyl groups for biomedical applications. J Biosci Bioeng, 2011. 112(5): p. 491-494.
17.Panouille, M. and V. Larreta-Garde, Gelation behaviour of gelatin and alginate mixtures. Food Hydrocolloids, 2009. 23(4): p. 1074-1080.
18.Choi, Y.S., et al., Studies on gelatin-containing artificial skin: II. Preparation and characterization of cross-linked gelatin-hyaluronate sponge. J Biomed Mater Res, 1999. 48(5): p. 631-9.
19.Zheng, J.P., et al., Gelatin/montmorillonite hybrid nanocomposite. I. Preparation and properties. Journal of Applied Polymer Science, 2002. 86(5): p. 1189-1194.
20.Sakai, S., I. Hashimoto, and K. Kawakami, Synthesis of an agarose-gelatin conjugate for use as a tissue engineering scaffold. J Biosci Bioeng, 2007. 103(1): p. 22-6.
21.Chiang, T.-Y., et al., Physicochemical properties and biocompatibility of chitosan oligosaccharide/gelatin/calcium phosphate hybrid cements. Materials Chemistry and Physics, 2010. 120(2-3): p. 282-288.
22.Sikorski, P., et al., Evidence for Egg-Box-Compatible Interactions in Calcium−Alginate Gels from Fiber X-ray Diffraction. Biomacromolecules, 2007. 8(7): p. 2098-2103.
23.Chung, Y.S., et al., Preparation of hydroxyapatite/poly(vinyl alcohol) composite fibers by wet spinning and their characterization. Journal of Applied Polymer Science, 2007. 106(5): p. 3423-3429.
24.Rhee, S.-H. and J. Tanaka, Effect of citric acid on the nucleation of hydroxyapatite in a simulated body fluid. Biomaterials, 1999. 20(22): p. 2155-2160.
25.Payne, R.G., et al., Development of an injectable, in situ crosslinkable, degradable polymeric carrier for osteogenic cell populations. Part 2. Viability of encapsulated marrow stromal osteoblasts cultured on crosslinking poly(propylene fumarate). Biomaterials, 2002. 23(22): p. 4373-80.
26.Payne, R.G., et al., Development of an injectable, in situ crosslinkable, degradable polymeric carrier for osteogenic cell populations. Part 1. Encapsulation of marrow stromal osteoblasts in surface crosslinked gelatin microparticles. Biomaterials, 2002. 23(22): p. 4359-4371.
27.Orive, G., et al., Cell encapsulation: promise and progress. Nat Med, 2003. 9(1): p. 104-7.
28.Kosaraju, S.L., A. Puvanenthiran, and P. Lillford, Naturally crosslinked gelatin gels with modified material properties. Food Research International, 2010. 43(10): p. 2385-2389.
29.Peter, M., et al., Preparation and characterization of chitosan–gelatin/nanohydroxyapatite composite scaffolds for tissue engineering applications. Carbohydrate Polymers, 2010. 80(3): p. 687-694.
30.Reinholz, G.G., et al., Bisphosphonates directly regulate cell proliferation, differentiation, and gene expression in human osteoblasts. Cancer Research, 2000. 60(21): p. 6001-7.
31.Bello, J., H.C.A. Riese, and J.R. Vinograd, Mechanism of Gelation of Gelatin. Influence of Certain Electrolytes on the Melting Points of Gels of Gelatin and Chemically Modified Gelatins. The Journal of Physical Chemistry, 1956. 60(9): p. 1299-1306.
32.Lau, M.H., J. Tang, and A.T. Paulson, Effect of polymer ratio and calcium concentration on gelation properties of gellan/gelatin mixed gels. Food Research International, 2001. 34: p. 8.